Plasma gelsolin (PG), the extracellular isoform of a six domain protein that has an intracellular counterpart, functions in blood to disassemble actin filaments as part of the actin scavenging system. D187 mutations in PG render domain 2 proteolytically labile and aggregation prone, leading to a gain of toxic function disease associated with the process of amyloidogenesis. We have discovered that the D187(N,Y) mutations prevent Ca2+ binding to domain 2, rendering plasma gelsolin susceptible to aberrant endoproteolysis by furin in the Golgi during secretion. Once outside the cell, the 68 kDa fragment is cleaved again by specific matrix metalloproteases (MMPs) including MT1-MMP (MMP14) affording 5 and 8 kDa fragments. The amyloidogenesis of these fragments is hastened by sulfated glycosaminoglycan components of the extracellular matrix (ECM) found in affected tissues. The overall goals of this work are to understand the amyloid pathological cascade and to develop an effective therapeutic strategy against the gelsolin amyloidoses. In Specific Aim 1, we will characterize the peptides comprising human amyloid to be sure we identify all MMPs involved, perform biophysical studies that will reveal the mechanism of gelsolin amyloidogenesis and elucidate how the oligosaccharides composing the ECM accelerate and possibly stabilize gelsolin amyloid. In Specific Aim 2, we will focus on developing and characterizing both cell-based and murine models of gelsolin familial amyloidosis (FAF). Cell-based models will allow us to characterize the trafficking pathways involved in FAF disease and understand compartmentalization of the pathological cascade. The FAF mouse model developed in the first funding period faithfully recapitulates many features of FAF pathology, including the intracellular inclusions also associated with inclusion body myositis (IBM), the most common muscle degenerative disease in the aging population. These valuable models will be used both to scrutinize our understanding of the FAF pathogenic cascade and to evaluate therapeutic strategies. In Specific aim 3, we will use the cell- and murine-based models of FAF to evaluate the potential of MMP inhibitors and small molecules that antagonize the ECM-gelsolin amyloid interaction to ameliorate FAF. The elucidation of the FAF pathogenic cascade together with the development of candidate pharmacologic agents will significantly advance our understanding of human amyloid diseases.